scholarly journals Duplex real-time PCR assay targeting insertion elements IS1081 and IS6110 for detection of Mycobacterium bovis in lymph nodes of cattle

2014 ◽  
Vol 30 (1) ◽  
pp. 45-59 ◽  
Author(s):  
A. Selim ◽  
M. El-Haig ◽  
W. Gaede
Keyword(s):  
Lymph Node ◽  
Lymph Nodes ◽  
Real Time ◽  
Mycobacterium Bovis ◽  
Real Time Pcr ◽  
Rapid Screening ◽  
Microscopical Examination ◽  
Pcr Assay ◽  
Specificity And Sensitivity ◽  
Insertion Elements

The development of a reliable and rapid screening test for detection of Mycobacterium bovis (M. bovis) helps in control of bovine tuberculosis. The aim of this study was evaluate a sensitive and specific assay for detecting M. bovis DNA in lymph nodes with lesions suggestive to tuberculosis taken from slaughtered cattle. A duplex real-time PCR assay was developed for the identification of M. bovis targeting insertion elements (IS) IS1081 and IS6110 in one internally controlled reaction. M. bovis DNA extraction protocols from tissue samples were evaluated. The specificity and sensitivity of the assay were evaluated for detecting serial dilutions of reference Mycobacteria strains as well as from spiked lymph node homogenate. Results revealed that microscopical examination of 600 lymph nodes with tuberculous-like lesion for detection of Acid-fast bacilli (AFB) showed a detection rate of 96.6%, compared to 98% M. bovis with duplex real-time PCR. The reproducible detection limit of the IS1081-PCR was 10 M. bovis cells/ml and the IS6110-PCR was 100 M. bovis cells/ml. Besides, both primer set of PCR protocol could detect 20 M. bovis cells/ml in spiked lymph node tissue. The assay was evaluated on 19 bacterial strains and was determined to be 100% specific for M. bovis. We suggest that the IS1081-PCR is a good candidate assay for routine screening of cattle lymph nodes and other tissue for M. bovis infection.

scholarly journals A New Highly Sensitive and Specific Real-Time PCR Assay Targeting the Malate Dehydrogenase Gene ofKingella kingaeand Application to 201 Pediatric Clinical Specimens

2018 ◽  
Vol 56 (8) ◽  
Author(s):  
Nawal El Houmami ◽  
Guillaume André Durand ◽  
Janek Bzdrenga ◽  
Anne Darmon ◽  
Philippe Minodier ◽  
...  
Keyword(s):  
Real Time ◽  
Malate Dehydrogenase ◽  
Real Time Pcr ◽  
Detection Sensitivity ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Content Type ◽  
Clinical Specimens ◽  
Specificity And Sensitivity ◽  
Pcr Assays

ABSTRACTKingella kingaeis a significant pediatric pathogen responsible for bone and joint infections, occult bacteremia, and endocarditis in early childhood. Past efforts to detect this bacterium using culture and broad-range 16S rRNA gene PCR assays from clinical specimens have proven unsatisfactory; therefore, by the late 2000s, these were gradually phased out to explore the benefits of specific real-time PCR tests targeting thegroELgene and the RTX locus ofK. kingae. However, recent studies showed that real-time PCR (RT-PCR) assays targeting theKingellasp. RTX locus that are currently available for the diagnosis ofK. kingaeinfection lack specificity because they could not distinguish betweenK. kingaeand the recently describedKingella negevensisspecies. Furthermore,in silicoanalysis of thegroELgene from a large collection of 45K. kingaestrains showed that primers and probes fromK. kingaegroEL-based RT-PCR assays display a few mismatches withK. kingae groELvariations that may result in decreased detection sensitivity, especially in paucibacillary clinical specimens. In order to provide an alternative togroEL- and RTX-targeting RT-PCR assays that may suffer from suboptimal specificity and sensitivity, aK. kingae-specific RT-PCR assay targeting the malate dehydrogenase (mdh) gene was developed for predicting no mismatch between primers and probe and 18 variants of theK. kingae mdhgene from 20 distinct sequence types ofK. kingae. This novelK. kingae-specific RT-PCR assay demonstrated high specificity and sensitivity and was successfully used to diagnoseK. kingaeinfections and carriage in 104 clinical specimens from children between 7 months and 7 years old.

scholarly journals Comparison data of a two-target real-time PCR assay with and without an internal control in detecting Salmonella enterica from cattle lymph nodes

Data in Brief ◽  
2018 ◽  
Vol 18 ◽  
pp. 1819-1824
Author(s):  
Jianfa Bai ◽  
Valentina Trinetta ◽  
Xiaorong Shi ◽  
Lance W. Noll ◽  
Gabriela Magossi ◽  
...  
Keyword(s):  
Lymph Nodes ◽  
Real Time ◽  
Internal Control ◽  
Real Time Pcr ◽  
Salmonella Enterica ◽  
Pcr Assay ◽  
Comparison Data

Relative accuracy, specificity and sensitivity of a 5′ nuclease Real-Time PCR assay for Salmonella detection in naturally contaminated pork cuts

2014 ◽  
Vol 28 (4) ◽  
pp. 133-137 ◽  
Author(s):  
Frédérique Pasquali ◽  
Alessandra De Cesare ◽  
Federica Bovo ◽  
Andrea Serraino ◽  
Gerardo Manfreda
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Relative Accuracy ◽  
Pcr Assay ◽  
Specificity And Sensitivity

scholarly journals Development of LAMP and Real-Time PCR Methods for the Rapid Detection of Xylella fastidiosa for Quarantine and Field Applications

2010 ◽  
Vol 100 (12) ◽  
pp. 1282-1288 ◽  
Author(s):  
S. J. Harper ◽  
L. I. Ward ◽  
G. R. G. Clover
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Color Change ◽  
Xylella Fastidiosa ◽  
Bacterial Species ◽  
Magnetic Bead ◽  
Endpoint Detection ◽  
Pcr Assay ◽  
Time Sensitivity ◽  
Specificity And Sensitivity

Xylella fastidiosa is a regulated plant pathogen in many parts of the world. To increase diagnostic capability of X. fastidiosa in the field, a loop-mediated isothermal amplification (LAMP) and real-time polymerase chain reaction (PCR) assay were developed to the rimM gene of X. fastidiosa and evaluated for specificity and sensitivity. Both assays were more robust than existing published assays for detection of X. fastidiosa when screened against 20 isolates representing the four major subgroups of the bacterium from a range of host species. No cross-reaction was observed with DNA from healthy hosts or other bacterial species. The LAMP and real-time assays could detect 250 and 10 copies of the rimM gene, respectively, and real-time sensitivity was comparable with an existing published real-time PCR assay. Hydroxynapthol blue was evaluated as an endpoint detection method for LAMP. When at least 500 copies of target template were present, there was a noticeable color change indicating the presence of the bacterium. Techniques suitable for DNA extraction from plant tissue in situ were compared with a standard silica-column-based laboratory extraction method. A portable PickPen and magnetic bead system could be used to successfully extract DNA from infected tissue and could be used in conjunction with LAMP in the field.

scholarly journals Molecular-Beacon Multiplex Real-Time PCR Assay for Detection of Vibrio cholerae

2006 ◽  
Vol 72 (9) ◽  
pp. 6424-6428 ◽  
Author(s):  
Aneta J. Gubala ◽  
David F. Proll
Keyword(s):  
Vibrio Cholerae ◽  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
Molecular Beacon ◽  
Environmental Water ◽  
Molecular Beacons ◽  
Pcr Assay ◽  
Specificity And Sensitivity ◽  
Pcr Assays

ABSTRACT A multiplex real-time PCR assay was developed using molecular beacons for the detection of Vibrio cholerae by targeting four important virulence and regulatory genes. The specificity and sensitivity of this assay, when tested with pure culture and spiked environmental water samples, were high, surpassing those of currently published PCR assays for the detection of this organism.

scholarly journals One-Tube Nested Real-Time PCR Assay for Rapid Screening of Porcine Cytomegalovirus in Clinical Samples

2020 ◽  
Vol 7 ◽  
Author(s):  
Hye-young Wang ◽  
Joong Ki Song ◽  
Seongho Shin ◽  
Ki Myung Choi ◽  
Hyunil Kim
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rapid Screening ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Porcine Cytomegalovirus

scholarly journals Multiplex real-time PCR assay combined with rolling circle amplification (MPRP) using universal primers for non-invasive detection of tumor-related mutations

RSC Advances ◽  
2018 ◽  
Vol 8 (48) ◽  
pp. 27375-27381 ◽  
Author(s):  
Jian Gong ◽  
Yishuai Li ◽  
Ting Lin ◽  
Xiaoyan Feng ◽  
Li Chu
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rolling Circle Amplification ◽  
High Specificity ◽  
Universal Primers ◽  
Multiplex Detection ◽  
Pcr Assay ◽  
Rolling Circle ◽  
Non Invasive ◽  
Specificity And Sensitivity

The MPRP system for SNP discrimination was developed, which showed high specificity and sensitivity for multiplex detection of tumor-related mutations.

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